1.3.2S
OVER-EXPRESSION OF HOST RESPONSE GENES: EFFECTS ON CELLULAR RESPONSES TO FUNGAL PATHOGENS

WR BUSHNELL, RW GIROUX, B BUCCIARELLI, AJ NELSON and LJ SZABO

US Department of Agriculture, Agricultural Research Service, Cereal Disease Laboratory, 1551 Lindig Street, University of Minnesota, St Paul MN 55108, USA

Background and objectives
An important method for demonstrating the role in host-parasite interaction of a given gene is to clone and introduce the gene into plants. Transgenic plants expressing the gene are then evaluated for alteration in host responses, pathogen development, or other aspects of the interaction. To facilitate such experiments, especially with cereals, which require time-consuming and expensive procedures to transform, we have developed a transient expression assay for rapid gene evaluation [1]. Gene effects are evaluated by microscopic examination of living, transiently expressing host cells in tissue inoculated with a pathogen or treated with toxins or elicitors.

Materials and methods
Coleoptiles from 4-7-day-old seedlings of oat or barley are split longitudinally and incubated on a mannitol-sorbitol osmoticum for 4 h or more. The adaxial epidermis is then bombarded with high-speed microparticles coated with DNA of two plasmids: one carrying a gene to be tested, and the other carrying maize C1 and R genes to mark individual cells that have a high probability of expressing the test gene. The C1 and R genes regulate anthocyanin synthesis. Cells expressing these genes turn red within 2 days. Rates of co-expression of anthocyanin and test genes are high. In trials with the uidA for GUS as test gene, 87% of red cells expressed GUS. 2-3 days after bombardment, tissues are inoculated with a pathogen [usually Blumeria (Erysiphe) graminis] or treated with toxin. Most genes tested so far code for proteins that are secreted outside protoplasts.

Results and conclusions
Genes for a rice chitinase, a maize ribosome-inhibiting protein, and an oat thaumatin-like protein (TLP) each reduced rates of infection (formation of haustoria by appressoria) by B. graminis by 48-78% of rates in controls, indicating the antifungal products of these genes strongly inhibited infection processes. Two TLP genes from oat [2] differed in activity: tlp4 was relatively inactive compared to tlp1, indicating that the transient expression assay can be used to detect differences in effectiveness of related genes. However, a mutant gene for an inactive form of glucanase reduced infection by 33%, suggesting that accumulation of secreted gene products in cell walls may contribute to infection rate reduction in ways unrelated to enzymatic or other antifungal toxicity. The transient expression assay was also used to detect a gene that confers sensitivity of resistant oat cells to the host-specific toxin, victorin. We tested four genes of the H-subunit protein of glycine decarboxylase, a victorin-binding protein [3], cloned by J. Lorang and T. Wolpert. One of the genes significantly increased rates of cell death in toxin-treated tissues normally resistant to the toxin, pinpointing the gene for further genetic and physiological evaluation of its role in victorin sensitivity. Overall, the transient expression assay is potentially useful for evaluating a wide range of diverse plant genes postulated to influence host-parasite interaction.

References
1. Nelson AJ, Bushnell WR, 1997. Transgenic Research 6, 233-244.
2. Lin K-C, Bushnell WR, Szabo LJ, Smith AG, 1996. Molecular Plant-Microbe Interactions 9, 511-522.
3. Wolpert TJ, Navarre DA, Moore DL, Macko V, 1994. Plant Cell 6, 1145-1155.